This proposal brings together four research teams with expertise in cancer genome sequencing, single-cell sequencing, and cancer cell biology to advance single-cell sequencing technology to tackle compelling questions in basic and translational cancer research. It responds to RFA-CA-17-023, Cancer Moonshot Initiative,andproposestocreateandvalidateaplatformofexperimentalandanalyticalapproachesforprecise single-cell sequencing of human samples based on the most recent advance in single-cell whole-genome amplification, Linear Amplification after Transposon Insertion (LIANTI), developed by the Xie laboratory. The immediate goal is to further develop LIANTI and augment bioinformatic analysis for single-cell LIANTI sequencing.
Aim1 focusesondevelopingahighthroughputversionofLIANTI.
Aim2 focusesondeveloping computational methods that utilize the digital and linear nature of LIANTI sequencing data.
Aim 3 describes strategiesandexperimentalmodelsthatwillbeusedtovalidateandbenchmarksingle-cellLIANTIsequencing performanceanddemonstrateitsapplicabilitytotumoranalysis.Overall,ourgoalistooptimizeandimplement the most precise single-cell sequencing technology for variant detection in single cancer or somatic cells. Successoftheprojectwillresultinanintegratedplatformthatovercomesprevailingimpedimentsinsingle-cell analysis and can have a transformative impact in multiple areas of cancer research, including tumor genetic heterogeneity, circulating tumor cells, primary/metastasis relationship, drug resistance, and benign-to- malignancytransformation.
We will further develop single-cell whole-genome amplification technologies, Linear Amplification after Transposon Insertion (LIANTI), and companion bioinformatic analysis pipelines. We will benchmark and validatethepotentialfordetectingsingle-nucleotidevariants,chromosomalrearrangements,andabsoluteDNA copy number in a single cell genome by LIANTI sequencing both using cancer cell lines and by live-cell experiments.
